Mucosal oncogenic human papillomaviruses (HPV) cause cervical cancer and likely cause a significant fraction of head and neck cancers. We and others have identified HPV-16 isolates that can establish persistent replication and immortalize primary human keratinocytes from various sites where HPV associated cancer arise. Assays we developed allow us to evaluate the effect of mutations on individual viral genes throughout the life cycle, including viral gene expression, initial DNA amplification, viral persistence and the influence of epithelial maturation on viral functions using raft cultures. These assays have allowed us to map the structure and regulation of the E1 cistron that is expressed from a novel promoter we have recently defined with a start site at nt 14 (P14). HPV-16 positive carcinomas of the cervix and the head and neck contain and express the viral E6-E7 oncogenic region from a promoter at nt 97 (P97). HPV DNA found in tumors and derived cell lines is frequently disrupted and integrated into the cellular genome and/or contains mutations. These alterations often disrupt the E2 gene, a critical regulator of gene expression. HPV DNAs isolated from carcinomas also frequently contain nucleotide sequence alterations in the viral upstream regulatory region. Our recent studies found these altered HPV genomes could lead to a more aggressive phenotype in our HPV dependent, cancer tissue culture model leading to increases in viral gene expression, virus replication and cell life span and growth rate. The questions this proposal addresses are: 1. What roles do alterations in the upstream regulatory region of HPVs isolated from head and neck cancers have in their pathogenesis? 2. How does E1 expression contribute to establishment of viral persistence? 3. How does E2 expression contribute to establishment of viral persistence?